Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or 12-0-tetradecanoyl-phorbol-13 acetate (TPA), which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration dependent manner when measured using in vitro enzyme assay with histone as substrate. This effect is mediated through nicotine cholinergic receptors since 1,1-dimethyl-phenyl- piperazinium (DMPP), a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]PdBu binding within one minute, the effect of which was maximal between 3 and 12 minutes. This effect was similarly reversed by chlorisondamine after 12 min and 18 hrs of nicotine treatment, indicating that continual nicotinic receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine stimulated diacylglycerol (DAG) production was slow, and it was observed after 12 min of incubation with nicotine. The DAG levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 hrs with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular weight of 82kD in the Western blot. These results suggest that nicotine activation increases particulate PKC activity and immunoreactivity in BAM cells. The long-term PKC activation may serve several functions, such as activation of mRNA production and a negative feedback regulation of either nicotinic receptors or voltage dependent Ca++-channels.